Many organisms employ a fascinating array of gene silencing pathways - including the best- known example, RNA interference (RNAi) - to confront invasive foreign nucleic acids. In 2007, a novel genetic interference pathway was discovered in bacteria and archaea. The clustered, regularly interspaced, short palindromic repeat (CRISPR) loci and their associated cas genes provide adaptive, inheritable, sequence-based genetic interference against viruses and mobile elements. Over the past 7 years, incredible progresses have been made in elucidating the functions and mechanisms of CRISPR-Cas as a defense system. However, the non-canonical functions of CRISPR-Cas components beyond defense, in areas like endogenous gene regulation and bacteria pathogenesis, were poorly understood and have only recently begun to be appreciated. We are using the gram-negative human pathogen Neisseria meningitidis (Nm) as a model system, because of its clinical importance and experimental tractability. Our preliminary findings prompted me to hypothesize that the meningococcal CRISPR-Cas9 system has alternative roles in post-transcriptional gene regulation and in bacterial physiology. The goal of this proposal is to test this hypothesis and to address some fundamental questions including: How does NmCas9 recognize and cleave RNA targets? What endogenous transcripts are regulated by CRISPR-Cas9? And, finally, how is Cas9 involved in meningococcal physiology and virulence? In the K99 phase of this award, I will perform in-depth analyses of the unusual RNA recognition and cleavage activities of NmCas9 in vitro. In addition, I will identify endogenous meningococcal RNAs that associate with, or are regulated by NmCas9. Moreover, I will also examine how is NmCas9 involved in adherence to human epithelial cells, and search for additional Cas9-dependent phenotypes in various aspects of meningococcal physiology and pathogenicity. With the biochemical skills developed and NmCas9's regulatory targets defined, I will further dissect and reconstitute NmCas9-based regulation of endogenous transcripts during the R00 phase. In addition, new NmCas9-dependent virulence defects identified in the K99 phase will be further characterized during the R00 phase. Taken together, my proposal will shed light on the understandings of alternative roles of CRISPR-Cas systems in gene regulation and bacterial physiology. Importantly, findings from this proposal will be of importance to a wide audience with interests in exploiting CRISPR-Cas9 systems in practical areas like antimicrobial treatments, human meningococcal disease interventions, as well as the developments of novel Cas9-based RNA targeting tools.